Acquired immunodeficiency syndrome (AIDS) is one of the most lethal diseases for which no complete cure has been identified. Basic research has attributed the cause of AIDS to a single-stranded RNA virus (retrovirus) referred to as human immunodeficiency virus (HIV) (Coffin, et al. (1986) Science 232:697; Gallo and Montagnier (1988) Sci. Am. 259:40). Two genetically distinct subtypes, HIV-1 and HIV-2 (Clavel, et al. (1986) Nature 324:691; Guyader, et al. (1987) Nature 326:662), have been recognized, with the former being identified as the main causative agent of the disease.
Reverse transcriptase is an essential enzyme necessary for HIV genomic replication (DeClerq (1986) J. Med. Chem. 29:1561-1569; Krug and Berger (1991) Biochemistry 30:10614-10623; Kedar, et al. (1990) Biochemistry 29:3603-3611). HIV reverse transcriptase is a multi-functional enzyme having RNA- and DNA-dependent DNA polymerase activity as well as a ribonuclease H (RNase H) activity. These activities enable the enzyme to reverse transcribe viral RNA to double-stranded DNA, hence fundamentally making it one of the most challenging central drug targets in anti-retroviral therapy (Gilboa, et al. (1979) Cell 18:93-100). In general, reverse transcriptase inhibitors fall into one of three classes: nucleoside inhibitors (NRTIs) which inhibit viral replication by acting as chain terminators of DNA synthesis; non-nucleoside inhibitors (NNRTIs), a structurally diverse class of compounds; and oligonucleotide constructs (ONRTI); however, most reverse transcriptase inhibitors primarily target the DNA polymerase activity and not the RNase H activity of this enzyme.
RNase H activity of HIV-1 reverse transcriptase is vital for viral replication since it is specifically required to cleave the RNA portion of a DNA/RNA heteroduplex intermediate, thereby permitting the viral DNA to disengage and invade the host cell's genetic material. Furthermore, point mutations in the RNase H domain of reverse transcriptase provoke a marked decrease in the level of virus proliferation, demonstrating that a functional RNase H activity is essential for retroviral replication (Mizrahi, et al. (1994) J. Biol. Chem. 269:19245-19249). HIV-1 RNase H inhibition has been demonstrated in vitro, however, it is unclear whether the inhibitory agents directly bind to the RNase H domain to achieve their effect (Tarrago-Litvak, et al. (2002) Current Pharmaceutical Design 8:595-614).
Blocking reverse transcriptase-associated RNase H activity has mostly been demonstrated in cell-free systems. For example, the RNase H activity of reverse transcriptase may be inhibited by 3′-azidothymidylate 5′-monophosphate (AZT-MP), a major intracellular metabolite of the NNRT inhibitor AZT, with an IC50 in the 50 μM range (Tan, et al. (1991) Biochemistry 30:4831-4835; Zhan, et al. (1994) Biochemistry 33:1366-1372). Apart from a high inhibitory concentration, the activity of AZT-MP is also dependent on the presence of a metal cation, with Mg2+ being the most effective co-activator.
The metal chelator N-(4-tert-butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazone (BBNH) has demonstrated potent RNase H inhibitory activity (IC50=3.5 μM), and is effective against mutant reverse transcriptase enzymes that have a high-level of resistance to other NNRTIs (Borkow, et al. (1997) Biochemistry 36:3179-3185). However, BBNH also inhibits the DNA polymerase activity of reverse transcriptase, and thus may interact with more than one domain on reverse transcriptase.
Illimaquinone, a natural product of marine origin, preferentially inhibits the RNase H activity of HIV-1 reverse transcriptase, however, it is not specific to HIV-1 as it also hinders the RNase H function of HIV-2 reverse transcriptase, MLV reverse transcriptase and E. coli (Loya and Hizi (1993) J. Biol. Chem. 268:9323-9328; Loya, et al. (1990) Antimicrob. Agents Chemother. 34(10):2009-12).
Few ONRTIs are specific for RNase H activity of HIV-1 reverse transcriptase. ONRTIs may act by blocking the catalytic site of the enzyme or impeding the binding of the viral DNA/RNA heteroduplex to the RNase H domain. Phosphorothioate oligonucleotides have demonstrated RNase H inhibition, however they also affect the DNA polymerase activity (Gao, et al. (1992) Mol. Pharmacol. 41:223-229). A series of DNA aptamers with high affinity and specificity for the RNase H activity of HIV-1 reverse transcriptase have also been isolated by SELEX. The most potent inhibitors were based on a G-quartet motif with IC50 values in the 500 nM range, however, these agents also inhibited the DNA polymerase activity of reverse transcriptase (Andreola, et al. (2001) Biochemistry 40:10087-10094). RNA aptamers also display non-selective dual inhibitory capacity (Chen and Gold (1994) Biochemistry 33:8746-8756). Duplexes consisting of 2′,5′-RNA/RNA have also been shown to competitively suppress binding of the viral DNA/RNA substrate to HIV-1 reverse transcriptase without evoking its RNase H activity (Wasner, et al. (1998) Biochemistry 37:7478-7486); however, the effect on the polymerase activity was not indicated.
Chimeric RNA/DNA oligonucleotides bearing a sense RNA and antisense DNA strand linked by two alkyl loop structures have been investigated for their ability to inhibit HIV replication (Park, et al. (2000) Biochem. Biophys. Res. Commun. 270(3) :953-60). Specifically, the constructs bear an antisense DNA oligonucleotide, complementary to the HIV-1 gag RNA sequence, which hybridizes to a complementary RNA oligonucleotide in the dumbbell structure. Upon delivery into the retrovirus-infected cells, cellular RNase H degrades the RNA portion of the dumbbell, thereby releasing the antisense DNA. The liberated antisense molecule then hybridizes to its complementary target viral RNA, thereby invoking RNase H-mediated degradation of the viral RNA strand. While effective at blocking viral proliferation, the mechanism of action of these chimeric dumbbells was designed to target viral gene expression using an antisense mechanism of action and not to inhibit a specific enzymatic function during HIV replication.
Circular dumbbell oligonucleotides have also demonstrated significant biological relevance as aptamers or decoys for hybridizing proteins such as transcription factors (Clusel, et al. (1993) Nucleic Acids Res. 21(15):3405-11; Lim, et al. (1997) Nucl. Acids Res. 25:575-581) and exhibit relatively high nuclease resistance as well as increased cellular uptake compared to their nicked and linear counterparts (Park, et al. (2000) supra; Yamakawa, et al. (1998) Bioorg. Med. Chem. 6(7):1025-32; Yamakawa, et al. (1996) Nucleosides & Nucleotides 15:519-529).
Accordingly, there is a need in the art to have reverse transcriptase RNase H inhibitors that exhibit high inhibitory activity and specificity against the RNase H activity of HIV-1 reverse transcriptase without interfering with polymerase function. Furthermore, it is desirable that such inhibitors of RNase H activity are specific for viral RNase H with minimal or no affinity for human ribonucleases.